Download MLDH serves as a sarcolemmal KATP channel subunit essential for

The EMBO Journal Vol. 21 No. 15 pp. 3936±3948, 2002
M-LDH serves as a sarcolemmal KATP channel
subunit essential for cell protection against ischemia
Russell M.Crawford, Grant R.Budas,
So®ja JovanovicÂ, Harri J.Ranki,
Timothy J.Wilson1, Anthony M.Davies and
Aleksandar JovanovicÂ2
Tayside Institute of Child Health, Ninewells Hospital & Medical
School, University of Dundee, Dundee DD1 9SY and
1
Division of Biological Chemistry & Molecular Microbiology,
School of Life Sciences, University of Dundee, Dundee DD1 4HN, UK
2
Corresponding author
e-mail: [email protected]
ATP-sensitive K+ (KATP) channels in the heart are
normally closed by high intracellular ATP, but are
activated during ischemia to promote cellular survival. These channels are heteromultimers composed
of Kir6.2 subunit, an inwardly rectifying K+ channel
core, and SUR2A, a regulatory subunit implicated in
ligand-dependent regulation of channel gating. Here,
we have shown that the muscle form (M-LDH), but
not heart form (H-LDH), of lactate dehydrogenase is
directly physically associated with the sarcolemmal
KATP channel by interacting with the Kir6.2 subunit
via its N-terminus and with the SUR2A subunit via its
C-terminus. The species of LDH bound to the channel
regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of
M-LDH in the channel protein complex was required
for opening of KATP channels during ischemia and
ischemia-resistant cellular phenotype. We conclude
that M-LDH is an integral part of the sarcolemmal
KATP channel protein complex in vivo, where, by
virtue of its catalytic activity, it couples the metabolic
status of the cell with the KATP channels activity that
is essential for cell protection against ischemia.
Keywords: heart/KATP channels/Kir6.2/lactate
dehydrogenase/SUR2A
Introduction
Sarcolemmal ATP-sensitive K+ (KATP) channels belong to
the group of intracellular ATP sensors, coupling the
metabolic status of the cell with the membrane excitability
(Noma, 1983). These channels are selectively permeable
to K+ ions and are closed by high intracellular ATP levels
(Terzic et al., 1995; Ashcroft and Gribble, 1998). It has
been suggested that the opening of sarcolemmal KATP
channels promotes survival of cardiomyocytes during
metabolic stress (Jovanovic and JovanovicÂ, 2001a; Light
et al., 2001). Ischemia opens sarcolemmal KATP channels
before a signi®cant fall in the level of intracellular ATP
occurs, suggesting that the intracellular level of ATP is not
the only factor that regulates the channel activity (Knopp
et al., 1999). Indeed, it is believed that the activity of KATP
3936
channels is controlled by a complex interaction of many
intracellular factors and signalling pathways. In addition to
ATP, the activity of these channels may be regulated by
other nucleotides, intracellular pH, lactate, cytoskeleton,
protein kinase C (PKC), phosphatidylinositol-4,5-bisphosphate and by the operative condition of the channel
itself (reviewed by Tanemoto et al., 2001).
Structurally, the cardiac subtype of KATP channels are
heteromultimers composed of the Kir6.2 subunit, an
inwardly rectifying K+ channel core primarily responsible
for K+ permeance, and SUR2A, a regulatory subunit
implicated in ligand-dependent regulation of channel
gating (Inagaki et al., 1996). More recently, evidence
has been provided that adenylate kinase and creatine
kinase, two main regulators of intracellular ATP levels in
the heart, are also parts of the cardiac KATP channel protein
complex (Carrasco et al., 2001; Crawford et al., 2002).
The whole channel protein complex seems to interact with
the actin cytoskeleton (Korchev et al., 2000). However,
the complexity of channel regulation would further
suggest the sarcolemmal KATP channel to be composed
of more proteins than those currently recognized.
In this regard, we have employed several strategies to
determine whether the sarcolemmal KATP channel protein
complex is composed of more proteins than Kir6.2,
SUR2A, adenylate kinase and creatine kinase. We have
found that the muscle form of lactate dehydrogenase
(M-LDH) is also an integral part of the sarcolemmal KATP
channel, where it regulates channel activity. Moreover, the
presence of M-LDH in the channel complex seems to be
required for early opening of KATP channels during
ischemia and cellular resistance against metabolic stress.
Results
Co-immunoprecipitation of the cardiac
membrane fraction
To immunoprecipitate proteins associated with the cardiac
KATP channels, we used anti-SUR2A antibody (see Ranki
et al., 2001; Crawford et al., 2002). Immunoprecipitation
revealed putative accessory polypeptides migrated at ~36
(p36), ~38 (p38), ~46 (p46), ~48 (p48), ~98 (p98) and
~150 kDa (p150) (Figure 1). The pro®le of immunoprecipitated peptides was different when anti-PKC antibody [nPKCe(n-17); Santa Cruz Biotechnology, Santa
Cruz, CA] was used instead of anti-SUR2A antibody
(Figure 1), and anti-SUR2A immunoprecipitation was
blocked with an excess of the corresponding antigenic
peptide (Figure 1).
LDH is a part of the sarcolemmal KATP
channel complex
Previously, p38, p48 and p150 were identi®ed as Kir6.2,
creatine kinase and SUR2A respectively (Crawford et al.,
ã European Molecular Biology Organization
M-LDH as a sarcolemmal KATP channel subunit
Fig. 1. Immunoprecipitation of cardiac membrane fraction. Coomassie
Blue stain of immunoprecipitate pellets (IP) obtained from cardiac
membrane fraction precipitated with either anti-SUR2A (without and
with antigenic peptide) or anti-PKC antibody.
2002). Matrix-assisted laser desorption/ionization time-of¯ight mass spectrometry (MALDI-TOF) analysis identi®ed p98, p46 and p36 as b-myosin, a-actin (data not
shown) and LDH (Figure 2A), respectively. LDH activity
was present in anti-SUR2A immunoprecipitation pellets,
whereas no activity was observed when the experimental
protocol was applied without the antibody (Figure 2B).
Western blotting with anti-LDH antibody (Abcam,
Cambridge, UK; this antibody targets all isoforms of
LDH) of both anti-Kir6.2 and anti-SUR2A immunoprecipitates revealed signals for LDH (Figure 2C).
Conversely, western blotting with anti-Kir6.2 and antiSUR2A antibodies detected Kir6.2 and SUR2A subunits
in anti-LDH immunoprecipitate of cardiac membrane
fraction (Figure 2D). Furthermore, double labelling
immuno¯uorescence showed that both Kir6.2 and
SUR2A co-localize with a substantial portion of LDH in
cardiomyocytes (Figure 3). This was observed in both rodshaped and rounded cells. In contrast, when cardiomyocytes were labelled with antibody against Kv 1.3, much
less spatial overlap was noted (Figure 3). To provide even
more evidence for physical association between LDH and
sarcolemmal KATP channels we performed ¯uorescence
resonance energy transfer (FRET) analysis using antibodies labelled with Alexa 594 (Molecular Probes,
Eugene, OR) (anti-LDH antibody, donor) and Alexa 647
(anti-Kir6.2 antibody, acceptor) applied on cardiac membrane fraction. The emission spectrum for this sample is
shown in Figure 4A, as are the donor and acceptor
components. The same data are presented in Figure 4B,
except that the magnitude of the extracted acceptor
spectrum is calculated from the (ratio)A observed for a
sample with only the anti-Kir6.2 antibody. Comparison of
the two panels shows that there is signi®cant energy
transfer between the ¯uorophores attached to the two
antibodies. The (ratio)A value is 0.246, one-third greater
than the value of 0.179 determined for the acceptor-only
control. SDS (1%), a disrupter of protein±protein complexes, abolished the observed energy transfer [Figure 4C;
(ratio)A decreased from 0.246 in the absence to 0.188 in
the presence of SDS, which was not signi®cantly different
from the acceptor-only control (0.192)]. When the same
type of experiment was carried out with anti-IgG antibody
Fig. 2. LDH co-immunoprecipitates with sarcolemmal cardiac KATP
channel protein complex and vice versa. (A) MALDI-TOF mass spectrum of tryptic mass ®ngerprint obtained from an ~36 kDa migrating
protein (identi®ed as LDH). (B) LDH assay with puri®ed LDH (B1)
and anti-SUR2A immunoprecipitate of cardiac membrane fraction
(B2). (C) Western blotting of anti-Kir6.2 and anti-SUR2A immunoprecipitate of cardiac membrane fraction with anti-LDH antibody.
(D) Western blotting of anti-LDH immunoprecipitate (IP) with antiKir6.2 and anti-SUR2A antibodies.
(Santa Cruz) in place of anti-Kir6.2 antibody, no energy
transfer was observed [Figure 4D; (ratio)A was 0.177 with
acceptor alone and 0.165 with acceptor±donor]. In
contrast, the energy transfer between anti-Kir6.2 (donor)
and anti-SUR2A (acceptor) antibodies was detected
[(ratio)A was 0.197 with acceptor alone and 0.213 with
acceptor±donor].
M-LDH, but not the heart form of LDH, is the
isoform directly physically associated with the
cardiac KATP channel subunits
LDH is a tetramer composed of either M (muscle) and/or
H (heart) subunits, which may be combined to form ®ve
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R.M.Crawford et al.
Fig. 3. Sarcolemmal KATP channel subunits, Kir6.2 and SUR2A, co-localize with LDH in cardiomyocytes. Original images of rod-shaped and rounded
cardiomyocytes stained with either anti-Kir6.2 or anti-SUR2A or anti-Kv 1.3 and anti-LDH antibodies labelled with rhodamine (red channel) or
¯uorescein (green channel) as indicated in the ®gure. Yellow colour is suggestive of co-localization. Scale bar, 45 mm.
LDH isozymes (Van Hall, 2000). We have co-expressed
Kir6.2 and/or SUR2A with M- and H-LDH subunits, and
examined which of the isozymes associate with the
channel subunits. When both H- and M-LDH subunits
were co-expressed with Kir6.2, SUR2A and Kir6.2/
SUR2A in A549 cells, cells natively devoid of KATP
channels, LDH activity was found in anti-Kir6.2 [antibody
published in Crawford et al. (2002), and it was used for
immunoprecipitation cells expressing Kir6.2 alone] and
anti-SUR2A (used for immunoprecipitation cells expressing Kir6.2 alone and Kir6.2/SUR2A) immunoprecipitates
of cells expressing subunits both alone and combined
(Figure 5A). However, when either H- or M-LDH was
co-expressed with channel subunits, LDH activity was
detected in immunoprecipitate from cells transfected with
M-LDH, but not in immunoprecipitate from cells transfected with H-LDH (Figure 5B and C). Western blotting
with the anti-LDH antibody of anti-Kir6.2 and antiSUR2A immunoprecipitates identi®ed the presence of
3938
LDH in cells transfected with Kir6.2, SUR2A, Kir6.2/
SUR2A and M-LDH, but not in those transfected with
H-LDH (Figure 5D and E). To determine whether M-LDH
is the only LDH subunit that associates with the KATP
channel subunits in vivo, we subjected anti-SUR2A
immunoprecipitate from cardiac membrane fraction to
two-dimensional (2D) gel analysis using an anti-LDH
antibody. 2D gel analysis revealed a signal corresponding
to M-LDH without any visible tracings of H subunits in
immunoprecipitate (Figure 5F).
M-LDH associates with Kir6.2 and SUR2A subunits
via its N- and C-termini, respectively
The results obtained with transfected A549 cells suggested
that M-LDH is associated with the KATP channel protein
complex interacting with both Kir6.2 and SUR2A subunits. Based on known LDH structure and primary
sequence differences between M- and H-LDH subunits
(see Discussion), we hypothesized that M-LDH interacts
M-LDH as a sarcolemmal KATP channel subunit
Fig. 4. Energy transfer between LDH and Kir6.2. (A) The emission spectra of LDH/Kir6.2, LDH donor control and Kir6.2 acceptor in PBS. (B) The
same as in (A) except that the magnitude of the extracted acceptor spectrum is calculated from the (ratio)A value from a sample with only the antiKir6.2 antibody. The difference between the sample data and calculated LDH/Kir6.2 spectrum (the sum of donor and acceptor spectra) shows energy
transfer. (C) The sample from (A) after the treatment with SDS (1%). (D) The emission spectra of LDH/IgG, LDH donor control and IgG acceptor in
PBS. AU, arbitrary units.
with Kir6.2 and SUR2A peptides via terminal ends. To test
this hypothesis, we made N- and C-terminus-truncated
forms of M-LDH (see Materials and methods). When an
N-terminus-truncated form of M-LDH was co-expressed
with the channel subunits in A549 cells, the activity
was lost only in immunoprecipitate from cells expressing Kir6.2 subunit alone (Figure 6A). Conversely, a
C-terminus truncated form of M-LDH was absent only in
immunoprecipitate of cells expressing the SUR2A subunit
alone (Figure 6A). The same pattern of LDH presence
was observed when western blotting with anti-LDH
antibody was applied instead of measuring LDH activity
(Figure 6B).
LDH substrates regulate sarcolemmal KATP
channels activity
The de®ning attribute of KATP channels is their inhibition
by intracellular ATP (Noma, 1983). Upon excision of a
membrane patch from guinea pig ventricular cardiomyo-
cyte, opening of KATP channels was inhibited by 1 mM of
ATP. Addition of pyruvate plus NADH (20 mM each) on
the intracellular face of the excised membrane patch
opened KATP channels despite the presence of ATP (1 mM;
Figure 7A). On the other hand, the opening of KATP
channels with 20 mM of lactate, in the presence of ATP
(1 mM), was inhibited by NAD (20 mM; Figure 7B).
Furthermore, we measured membrane currents from a
guinea pig ventricular myocyte in a whole-cell con®guration. When NADH (20 mM) or pyruvate (20 mM) was
present in the patch pipette solution alone, the steady-state
current±voltage relationship (Figure 8A) was in an N
shape due to the strong inward recti®cation of Ik1 channels
and absence of active KATP channels (Terzic et al., 1995).
However, when pyruvate (20 mM) was combined with
NADH (20 mM), the outward K+ current became greater
and inward recti®cation weaker (Figure 8A). The current
density was 6.6 6 0.8 and 7.5 6 0.8 pA/pF in the
presence of NADH and pyruvate alone, respectively, and
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R.M.Crawford et al.
Fig. 5. The muscle, but not heart, form of LDH directly associates with Kir6.2 and SUR2A subunits. (A±C) LDH assay with anti-SUR2A or antiKir6.2 immunoprecipitate of untransfected A549 cells or cells transfected with Kir6.2 and SUR2A (alone and in combination) plus M-LDH plus
H-LDH (A), H-LDH (B) or M-LDH (C). Anti-Kir6.2 antibody was used for immunoprecipitation of Kir6.2 cells, while for all other groups antiSUR2A antibody was applied. (D) Western blotting of anti-Kir6.2 and anti-SUR2A immunoprecipitate under conditions in (C). (E) Western blotting
of anti-Kir6.2 and anti-SUR2A immunoprecipitate under conditions in (B). (F±H) Western blotting 2D gel analysis with anti-LDH antibody of
anti-SUR2A immunoprecipitate (IP) of cardiac membrane fraction or puri®ed M- and H-LDH [Sigma; (G) and (H)]. Hollow circle in 2D gel in (F)
indicates the expected position for H-LDH signal.
Fig. 6. M-LDH associates with Kir6.2 and SUR2A subunits via N- and C-terminus, respectively. (A) LDH assay with anti-SUR2A or anti-Kir6.2
immunoprecipitate of A549 cells transfected with Kir6.2 and SUR2A (alone and in combination) plus N(DN)- or C(DC)-termini deletion mutants of
M-LDH. (B) Western blotting with anti-LDH antibody in conditions under (A).
3940
M-LDH as a sarcolemmal KATP channel subunit
control conditions and 28.2 6 3.1 pA/pF following
ischemia, P = 0.007, n = 5, Figure 9].
The opening of recombinant KATP channels as well
as intact LDH activity is required for cell protection
afforded by Kir6.2/SUR2A/M-LDH
Fig. 7. LDH substrates regulate the opening of KATP channels in
excised membrane patches. Recording of KATP channel activity in
membrane patch treated with ATP (1 mM) alone, ATP (1 mM) plus
pyruvate (20 mM) plus NADH (20 mM), again with ATP (1 mM)
alone and again with ATP (1 mM) plus pyruvate (20 mM) plus NADH
(20 mM) (A) or treated with ATP (1 mM), ATP (1 mM) plus lactate
(20 mM) and ATP (1 mM) plus lactate (20 mM) plus NAD (20 mM)
(B). Holding potential ±60 mV. Dotted lines correspond to the zero
current levels. Both types of experiment were repeated four times with
essentially similar results.
11.4 6 1.2 pA/pF in the presence of NADH plus pyruvate
(P = 0.01, n = 6±9; Figure 8A1). Lactate (20 mM)induced outward K+ current signi®cantly increased at
potentials more positive than ±70 mV, and inward
recti®cation of the current±voltage relationship was
much weaker (Figure 8B), which is a typical ®nding
when measuring K+ current ¯ows through KATP channels
(Terzic et al., 1995). When NAD (20 mM) was also
present in the pipette solution with lactate (20 mM), the
outward current was decreased and inward recti®cation
was stronger (Figure 8B). The current density was
6.8 6 1.0 and 18.1 6 3.2 pA/pF in the presence of
NAD and lactate alone, respectively, and 10.2 6 1.8 pA/
pF in the presence of NAD plus lactate (P = 0.019,
n = 6±9; Figure 8B1).
Kir6.2/SUR2A/M-LDH confer resistance to
ischemia in A549 cells
At rest, A549 cells, which are natively devoid of KATP
channels, have a low cytosolic Ca2+ concentration (30.2 6
5.7 nM, n = 16; Figure 9A). Exposure to ischemia
produced rapid and signi®cant Ca2+ loading (208.5 6
71.1 nM, n = 16, P = 0.03; Figure 9), which was associated with statistically non-signi®cant decrease in wholecell K+ current as measured by perforated patch±clamp
electrophysiology (current density at +80 mV was 21.9 6
1.9 pA/pF in control and 15.0 6 2.6 pA/pF in ischemia,
n = 6, P = 0.13; Figure 9). Transfection of Kir6.2/
SUR2A/M-LDH almost completely prevented ischemiainduced Ca2+ loading (resting Ca2+ was 55.6 6 9.0 nM
prior to and 73.3 6 10.8 nM following ischemia,
P = 0.22, n = 9; Figure 9). In transfected cells, ischemia
also induced signi®cant increase in whole-cell K+ membrane current as measured by perforated patch±clamp
electrophysiology [in this mode intracellular milieu
remains largely undisturbed (Jovanovic et al., 2001);
current density at +80 mV was 14.7 6 2.0 pA/pF under
The resistance of Kir6.2/SUR2A/M-LDH A549 phenotype
to ischemia was signi®cantly reduced when glybenclamide
(10 mM), a KATP channel blocker, was present during the
experiments (intracellular Ca2+ was 31.2 6 5.3 nM prior
to and 96.4 6 31.6 nM following ischemia, P = 0.008,
n = 9; Figure 10). In addition, glybenclamide (10 mM)
abolished the ischemia-induced increase in whole-cell
membrane K+ current (current density at +80 mV was
23.1 6 2.9 pA/pF in control and 24.2 6 3.2 pA/pF in
ischemia, n = 5, P = 0.63; Figure 10). When Kir6.2/
SUR2A were co-transfected with the inactive, mutated
form of M-LDH (His193 was mutated into glycine, which
greatly reduced the catalytic activity; Clarke et al., 1986)
instead of wild-type enzyme, the protection against
ischemia was abolished (intracellular Ca2+ was 37.0 6
6.5 nM prior to and 193.8 6 62.7 nM following ischemia,
P = 0.026, n = 6; Figure 10), as well as ischemia-induced
increase in whole-cell membrane K+ current (current
density at +80 mV was 20.1 6 3.1 pA/pF in control
and 19.3 6 2.9 pA/pF in ischemia, n = 5, P = 0.32;
Figure 10).
Discussion
It has recently been suggested that sarcolemmal KATP
channel complexes are composed of Kir6.2 and SUR2A
subunits as well as two enzymes, regulators of ATP levels,
adenylate kinase and creatine kinase (Inagaki et al., 1996;
Carrasco et al., 2001; Crawford et al., 2002). In the present
study, Coomassie Blue staining of the anti-SUR2A
immunoprecipitate revealed polypeptides that were previously identi®ed as Kir6.2, SUR2A and creatine kinase
(Crawford et al., 2002). The presence of adenylate kinase
in immunoprecipitate was not visualized, which is plausible since the molecular weight of adenylate kinase would
overlap with the primary antibody heavy chain under the
conditions applied (Carrasco et al., 2001). MALDI-TOF
analysis identi®ed p46 and p98 as a-actin and b-myosin,
which is in accord with the cardiac KATP channel protein
complex being physically associated with the cytoskeleton
(Korchev et al., 2000).
Several lines of evidence, including MALDI-TOF, LDH
assay and western blotting, suggested that a previously
unidenti®ed 36 kDa protein found in the immunoprecipitate was LDH. Since immunoprecipitations could have
non-speci®c contaminants (Harlow and Lane, 1999), we
used extreme care to classify LDH as a protein associated
with the sarcolemmal KATP channel complex. The
following results suggest that the LDH presence in
immunoprecipitate is due to the physical association
with KATP channel subunits: (i) the appearance of p36
was speci®c for the anti-SUR2A antibody and was blocked
by the antigenic peptide, suggesting that interaction
between the anti-SUR2A antibody and membrane fraction
is required for the presence of LDH in the immunoprecipitate; (ii) there was no cross-reactivity between the antiSUR2A antibody and puri®ed LDH itself, which excludes
3941
R.M.Crawford et al.
Fig. 8. LDH substrates regulate KATP channel-mediated membrane current. (A) Membrane currents and corresponding current±voltage relationships in
cells ®lled with pipette solution containing NADH (20 mM) or pyruvate (20 mM) or both. Arrow points to the zero current level. (A1) Current
densities at 80 mV for conditions in (A). Vertical bars represent mean 6 SEM (n = 6±9). *P <0.01. (B) Membrane currents and corresponding
current±voltage relationships in cells ®lled with pipette solution containing NAD (20 mM) or lactate (20 mM) or both. Arrow points to the zero current
level. In all experiments depicted cells were dialysed for ~5 min. (B1) Current densities at 80 mV under conditions in (B). Vertical bars represent
mean 6 SEM (n = 6±9). *P <0.05.
the possibility that LDH was directly precipitated with the
anti-SUR2A antibody; (iii) LDH was detected in immunoprecipitates obtained with structurally unrelated antibodies
against two members of the protein complex, Kir6.2 and
3942
SUR2A subunits; and (iv) not only was LDH found in both
anti-SUR2A and anti-Kir6.2 immunoprecipitates but, vice
versa, both SUR2A and Kir6.2 subunits were identi®ed in
anti-LDH immunoprecipitate of cardiac membrane frac-
M-LDH as a sarcolemmal KATP channel subunit
Fig. 9. Kir6.2/SUR2A/M-LDH confers resistance against ischemia in A549 cells. Original frames and whole-cell recordings under control and
ischemic conditions with corresponding bar (intracellular Ca2+ levels, frames) and scatter-line graphs (whole-cell recordings) of (A) untransfected
Fura-2 loaded A549 cells and (B) transfected with Kir6.2/SUR2A/M-LDH. Horizontal bar represents 20 mm. Vertical bars represent mean 6 SEM,
whereas points represent the mean (n = 6±16). *P <0.05.
tion. Immuno¯uorescence and FRET analysis were also
employed to test the conclusions drawn with immunoprecipitation data. Immuno¯uorescence demonstrated
co-localization of Kir6.2 or SUR2A subunits with LDH
in both rod-shaped and rounded cardiomyocytes, i.e. in
both healthy and dead cells, respectively (see S.JovanovicÂ
et al., 1998). In healthy cardiomyocytes, it could be argued
that LDH might appear to be co-localized with channel
subunits due to ubiquitous intracellular presence of the
enzyme. However, since dead cardiac cells release a major
fraction of intracellular LDH (Altschuld et al., 1994), this
argument does not stand, suggesting that the observed colocalization is speci®c for LDH and the channel subunits.
This conclusion is supported by ®nding that much less
spatial overlap was observed between Kv 1.3, another K+
channel subunit expressed in the heart (England et al.,
1995), and LDH. In addition, FRET analysis detected
energy transfer between Kir 6.2 and LDH in cardiac
3943
R.M.Crawford et al.
Fig. 10. The opening of KATP channels and intact activity of M-LDH is required for Kir6.2/SUR2A/M-LDH-mediated resistance. Original frames and
whole-cell recordings under control and ischemic conditions with corresponding bar (intracellular Ca2+ levels, frames) and scatter-line graphs (wholecell recordings) of A549 cells transfected with Kir6.2/SUR2A/M-LDH in the presence of 10 mM glybenclamide (A) and transfected with Kir6.2/
SUR2A plus 193gly-M-LDH (B) (single-amino acid mutated form of M-LDH with greatly reduced LDH activity; glybenclamide was absent). Scale
bar, 20 mm. Vertical bars represent mean 6 SEM; points represent mean (n = 5±9). *P <0.05.
membrane fraction suggesting that these two proteins are
in close proximity (see Clegg, 1992). As the Ro for this
donor±acceptor pair is 8.4 nm (see the manufacturer's web
site: www.probes.com), the average distance between the
¯uorophores must be <17 nm, as neglible FRET is
observed at distances greater than twice the Ro. Bearing
in mind that this distance includes a sum of anti-Kir6.2
antibody, Kir6.2 protein, LDH protein and anti-LDH
antibody lengths, it is very unlikely that in pure membrane
fraction vigorously cleaned of cytosolic elements Kir6.2
and LDH protein could be so close without being
3944
physically associated. This conclusion is supported by
the ®nding that treatment which disrupts protein complexes abolished the energy transfer between Kir6.2 and
LDH, while no energy transfer was observed between
LDH and IgG antibodies. Taken all together, it reasonable
to conclude that LDH is an integral part of the
sarcolemmal KATP channel protein complex in vivo.
LDH is a tetramer composed of M (muscle) and/or H
(heart) subunits, which may be combined to form ®ve
LDH isozymes. In the heart, all LDH isozymes are present;
LDH1 (H4) is the predominant form, whereas LDH5 (M4)
M-LDH as a sarcolemmal KATP channel subunit
is present in trace amounts (Van Hall, 2000). When H- and
M-LDH subunits in combination and alone were coexpressed with Kir6.2, SUR2A and Kir6.2/SUR2A in
A549 cells, the pattern of LDH presence in anti-Kir6.2 and
anti-SUR2A immunoprecipitate suggested that M-LDH,
but not H-LDH, is the isoform that associates with the
channel subunits. Since M- and H-LDH subunits are
indistinguishable by size but have different isoelectric
points (Li, 1990; Read et al., 2001; this study), we applied
isoelectric focusing and 2D gel analysis to determine
whether M-LDH is the only LDH subunit that associates
with the KATP channel subunits in vivo. This technique
revealed a signal corresponding to M-LDH without any
visible tracings of H subunits in immunoprecipitate,
con®rming the data obtained with the heterologues
expression system and suggesting that M-LDH is indeed
a part of the cardiac KATP channel complex in vivo. LDH is
composed of 331 amino acids, with the active site
localized approximately in the middle of the protein (Li,
1990; Van Hall, 2000). In order to be associated with both
Kir6.2 and SUR2A subunits and still retain its activity,
interacting parts of M-LDH with channel subunits probably have to be localized on its terminal sides. In addition,
differences in amino acid sequence between M-LDH
(associates with the channel) and H-LDH (does not
associate with the channel) are found primarily in the
®rst 22 and the last 38 residues (Li, 1990), again
suggesting that termini are involved in interaction between
M-LDH and Kir6.2/SUR2A. To test this hypothesis, we
made N- and C-terminus-truncated forms of M-LDH.
When an N-terminus-truncated form of M-LDH was coexpressed with the channel subunits in A549 cells, LDH
was lost only in immunoprecipitate from cells expressing
Kir6.2 subunit alone, while a C-terminus-truncated form
of M-LDH was absent only in immunoprecipitate of cells
expressing SUR2A subunit alone. These results suggest
that M-LDH associates with the Kir6.2 subunit via its
N-terminus and with the SUR2A via its C-terminus.
Although LDH is normally a tetramer (Li, 1990; Takatani
et al., 2001), the catalytic function of LDH does not
depend upon interaction between LDH subunits, and
single subunits retain catalytic activity (King and Weber,
1986). This means that the complex of Kir6.2, SUR2A and
M-LDH subunits may be functional even if LDH subunits
do not interact with each other. Also, since a single KATP
channel is composed of four Kir6.2 and four SUR2A
subunits (Shyng and Nichols, 1997), it is possible that four
M-LDH subunits associated with the channel subunits
form a three-dimensional structure that has a spatial
relationship similar to those in the traditional enzyme
tetramer without necessarily being physically associated.
It is generally accepted that M-LDH primarily controls
the formation of lactate (Van Hall, 2000). Lactate is
reported to open sarcolemmal KATP channels in cells
despite the presence of millimolar concentrations of ATP
(Han et al., 1993). Since LDH catalyses NADH + pyruvate
« lactate + NAD reaction, this enzyme has the potential
both to open (by lactate production) and to close (by
lactate removal) KATP channels. The results obtained, at
both single channel and whole-cell levels, demonstrate
that LDH substrates activate or inhibit KATP channels
opening in a manner consistent with the hypothesis that
LDH is a part of the sarcolemmal KATP channels. The
regulation of KATP channel activity was observed despite
the continuous intracellular presence of millimolar concentrations of ATP, suggesting that M-LDH, by virtue of
its activity, could open KATP channels at physiological
concentrations of ATP. In the heart, KATP channels are
opened during ischemia and act in a cardioprotective
manner (Knopp et al., 1999; Light et al., 2001). Although
Kir6.2/SUR2A reconstitutes basic properties of sarcolemmal KATP channels in cell lines natively devoid of
these channels (Inagaki et al., 1996), recombinant KATP
channels remain mostly closed throughout the metabolic
challenge (Jovanovic and JovanovicÂ, 2001c). Yet, when
recombinant channels are pharmacologically open host
cells are protected against metabolic stress (A.JovanovicÂ
et al., 1998; Jovanovic et al., 1999). Inability of Kir6.2/
SUR2A proteins to create cellular phenotype resistant to
metabolic stress may be due to incomplete reconstitution
of the channel and impaired metabolic sensing. We tested
the hypothesis that M-LDH, as a part of the sarcolemmal
KATP channel complex, may be important for co-ordinated
channel opening and cellular protection. A549 cells
responded to ischemia with intracellular Ca2+ loading,
which is a major indicator of the cell injury (A.JovanovicÂ
et al., 1998), showing that these cells are vulnerable to
such an insult. When Kir6.2/SUR2A were co-expressed
with M-LDH in A549 cells, ischemia-induced Ca2+
increase was almost abolished and this was associated
with signi®cant activation of the outward K+ current. Both
increase in K+ current and resistance towards ischemia
was inhibited by glybenclamide, an antagonist of KATP
channels, suggesting that the opening of recombinant
KATP channels was responsible for the observed cellular
resistance to ischemia. When Kir6.2/SUR2A was cotransfected with the inactive, mutated form of M-LDH,
protection against ischemia was abolished, as well as
ischemia-induced increase in whole-cell membrane K+
current, implying that the presence of M-LDH with
unimpaired activity is required in the cardiac KATP
channel protein complex for early opening of KATP
channels, clamping the membrane potential and maintaining the intracellular Ca2+ homeostasis.
M-LDH as a part of the cardiac KATP channel could
explain how the channel open during ischemia to protect
the cells despite the presence of millimolar levels of
ATP. Under anaerobic conditions, pyruvate, NAD and H+
cannot be metabolized further and accumulate (see Kantor
et al., 2001), leading to the M-LDH-catalysed lactate
production within microenvironment surrounding the
channel. Lactate opens the channel despite high levels of
ATP (Han et al., 1993; this study) and protects the cell
against Ca2+ overload and death.
In conclusion, we have demonstrated that M-LDH is an
integral part of the sarcolemmal KATP channel protein
complex in vivo, where it couples the metabolic status of
the cell with the KATP channels activity which, in turn, is
essential for cell protection against ischemia.
Materials and methods
Cardiac cells isolation
Ventricular cardiomyocytes were dissociated from guinea pig hearts as
described previously (Ranki et al., 2001). In brief, hearts were
retrogradely perfused (at 37°C) with medium 199, followed by Ca2+-
3945
R.M.Crawford et al.
EGTA-buffered low-Ca2+ medium (pCa = 7), and ®nally low-Ca2+
medium containing pronase E (8 mg/100 ml), proteinase K (1.7 mg/
100 ml), bovine serum albumin (BSA; 0.1 g/100 ml, fraction V) and
200 mM CaCl2. Ventricles were cut into fragments in the low-Ca2+
medium enriched with 200 mM CaCl2. Single cells were isolated by
stirring the tissue (at 37°C) in a solution containing pronase E and
proteinase K supplemented with collagenase (5 mg/10 ml).
A549 cells, genes, truncations, site-directed mutagenesis
and transfection
A549 cells (ATCC), which are natively devoid of KATP channels, were
cultured in a tissue ¯ask (at 5% CO2) containing Dulbecco's modi®ed
Eagle's medium (DMEM) supplemented with 10% fetal calf serum and
2 mM glutamine. Cells were transfected using 8±24 ml lipofectAMINE
(Gibco), with 2±6 mg of total plasmid DNA [full-length Kir6.2, SUR2A,
M-LDH, H-LDH, DN-LDH, DC-LDH, gly193-M-LDH cDNA and green
¯uorescent protein (GFP), subcloned into the mammalian expression
vector pcDNA3.1+]. Kir6.2 was a gift from Dr S.Seino (Chiba University,
Chiba, Japan) and SUR2A was kindly provided by Dr Y.Kurachi (Osaka
University, Osaka, Japan). H- and M-LDH were cloned from mouse heart
RNA. First strand cDNA was synthesized from 1 mg total RNA using
200 U of MML-V reverse transcriptase (Promega) and an anchoroligo(dT) [5¢-GTCATGGCATGGGATCCTG(T)15; total volume, 20 ml],
according to the manufacturer's instructions. The total PCR volume was
25 ml (50 ml for H-LDH), including 3 ml of the cDNA, 25 pmol (20 pmol
for H-LDH) of each primer and 2.5 U PromegaTag (Promega). PCR was
performed in a thermal cycler Model Phenix (Helena Biosciences) or a
thermal cycler Mastercycler Gradient (Eppendorf) under the following
conditions: the PCR was run with a hot start for 5 min at 95°C (initial
melting), followed by 40 cycles of 0.5 min at 94°C, 0.5 min at 55°C and
1.5 min at 72°C (®nal extension). The primers had the following
sequences. H-LDH-speci®c primers: sense, 5¢-GACAAGATGGCAACCCTTAAGGA-3¢; antisense, 5¢-AGTGCAAACATCAAACAAGCCTGG3¢. M-LDH-speci®c primers: sense, 5¢-ATGGCAACCCTCAAGGACCAG-3¢; antisense, 5¢-GCACAGCTGTGGGGAGTG-3¢. The product
obtained (full-length cDNA), which contain translatable parts of H-LDH
or M-LDH sequence, was isolated from a 1% agarose gel using Qiagen
gel extraction kit, sequenced, ligated into the pcDNA3.1+ mammalian
expression vector and used for transfection. Truncation of the ®rst 19
N-terminal amino acids of M-LDH was achieved using nested PCR of MLDH cDNA and the following primers: sense: 5¢-GAGCAGGCTCCCCAGAACAAGATTACAGTTATG-3¢; antisense, 5¢-GCAGAGCTGTGGGGAGTG-3¢. The total PCR volume was 50 ml, including 3 ml of
the cDNA, 20 pmol of each primer and 2.5 U PromegaTag (Promega).
PCR was performed in a thermal cycler Mastercycler Gradient
(Eppendorf) under the following conditions: the PCR was run with a
hot start for 5 min at 94°C (initial melt), followed by for 35 cycles of 10 s
at 94°C, 1.5 min at 56°C and 1.5 min at 71°C (®nal extension). Truncation
of 48 C-terminal amino acids was achieved by PCR using site-directed
mutagenesis to introduce a 3¢ stop codon (TAG). The QuikChange sitedirected mutagenesis kit (Stratagene) was used according to the
manufacturer's instructions. Mutagenic primers were sense: 5¢-TGATTAAGGGTCTCTATGGAATCAATGTAGATGTCTTCCTCAGTGTCCCATGTATCCTG-3¢; antisense, 5¢-CAGGATACATGGGACACTGAGGAAGACATCTACATTGATTCCATAGAGACCCTTAATCA-3¢.
PCR conditions were the same as for N-terminus truncation.
For site-directed mutagenesis of His193, the same Stratagene kit as for
C-terminus truncation was used. Mutagenic primers were sense: 5¢GCTGGGTCCTGGGAGAAGGTGGCGACTCC-3¢; antisense, 5¢-GGAGTCGCCACCTTCTCCCAGGACCCAGC-3¢. The total PCR volume
was 50 ml, including 25 ng of non-mutated M-LDH inserted into the
pcDNA3.1+, 125 ng of each primer, 1 ml of dNTP and 2.5 U DNA
polymerase. PCR was performed in a thermal cycler Model Phenix
(Helena Biosciences) under the following conditions: the PCR was run
with a hot start for 0.5 min at 95°C (initial melting), followed by 16 cycles
of 0.5 min at 95°C, 1 min at 55°C and 14 min at 68°C. The parental DNA
was destroyed according to the manufacturer's instructions, and the
mutated plasmids were further transformed, sequenced and used for
experiments. All PCR products in experiments described above were
veri®ed by DNA sequencing.
Immunoprecipitation, Coomassie Blue staining and western
blot analysis
Sheep anti-peptide antibodies were raised against synthetic peptides
conjugated to a carrier protein, keyhole limpet hemocyanin, and used for
immunoprecipitation and western blotting (Crawford et al., 2002).
Cardiac membrane fraction was obtained as described previously
3946
(Crawford et al., 2002). In brief, guinea pig ventricular tissue was
homogenized in buffer I [Tris 10 mM, NaH2PO4 20 mM, EDTA 1 mM,
phenylmethylsulfonyl ¯uoride (PMSF) 0.1 mM, pepstatin 10 mg/ml,
leupeptin 10 mg/ml pH 7.8] and incubated for 20 min (at 4°C). The
osmolarity was restored with KCl, NaCl and sucrose and the obtained
mixture was centrifugated at 500 g. The supernatant was diluted in buffer
II (imidazole 30 mM, KCl 120 mM, NaCl 30 mM, NaH2PO4 20 mM,
sucrose 250 mM, pepstatin 10 mg/ml, leupeptin 10 mg/ml pH 6.8) and
centrifugated at 7000 g, the pellet removed and the supernatant was
centrifugated at 30 000 g. The obtained pellet contains membrane
fraction. A549 cells or membrane fraction were snap-frozen and ground
to a powder under liquid nitrogen. The powder was resuspended in 10 ml
of buffer (20 mM HEPES, 150 mM NaCl, 1% Triton X-100 pH 7.5) and
homogenized. Protein concentration was determined using the method of
Bradford. Forty micrograms of the epitope-speci®c anti-SUR2A or antiKir6.2 antibody was pre-bound to protein G±Sepharose beads and used to
immunoprecipitate from 60 mg of protein extract. The pellets of this
precipitation were run on two SDS polyacrylamide gels, one for
Coomassie Blue stain and one for western blot analysis as described in
Crawford et al. (2002). In a separate series of experiments, immunoprecipitation was performed with the anti-LDH antibody (Abcam) using
the same protocol as described above. For the 2D gel analysis,
immunoprecipitation was performed using 20 mg of SUR2A speci®c
anti-peptide antibody to precipitate from 20 mg guinea pig cardiac
membrane fraction. The resultant precipitate was run on a Novex
(Invitrogen) isoelectric focusing gel encompassing a pH range of 3±10,
followed by excision of the lane and further protein separation on the
second dimension according to molecular weight. Control samples were
puri®ed H-LDH and H-LDH from Sigma run using the same protocol
(Crawford et al., 2002).
Immuno¯uorescence and laser confocal microscopy
Cardiomyocytes were ®xed with 4% paraformaldehyde for 20 min. After
washing with phosphate-buffered saline (PBS), cells were permeabilized
with 0.1% Triton X-100 in PBS for 15 min, washed and blocked with 5%
BSA in PBS for 30 min. Cells were incubated overnight at 4°C with
primary antibodies (rabbit anti-Kir6.2, rabbit anti-SUR2A, goat anti-LDH
and rabbit anti-Kv1.3) in PBS and incubated in the dark with the
secondary antibodies [anti-sheep ¯uorescein (Diagnostic Scotland,
Edinburgh, UK) and anti-rabbit rhodamine (Santa Cruz)] at 1:500
dilution for 1 h. Samples were washed with PBS, mounted on slides using
50% glycerol containing 1% n-propylgallate and sealed with nail polisher
(Marks & Spencer, Dundee, UK). Slides were examined by laser confocal
microscopy (LSM-510; Zeiss, Gottingem, Germany). Images were
acquired and analysed using Zeiss Image Examiner Software.
FRET
Cardiac membrane fraction was obtained as described in the immunoprecipitation section. Anti-Kir6.2, anti-LDH, anti-SUR2A or anti-IgG
antibodies were labelled with Alexa 594 (donor) or Alexa 647 (acceptor)
where appropriate, according to the manufacturers instruction (Molecular
Probes). Cardiac membrane fraction was blocked with 1% BSA,
incubated with labelled antibodies for 12 h at 4°C, pelleted, and the
pellets washed and resuspended with PBS. Fluorescence spectra were
recorded at 20°C using an SLM-Aminco 8100 ¯uorimeter, in PBS buffer
except where otherwise speci®ed. The excitation and emission band
passes were 4 and 8 nm, respectively. Scattering of incident light was
minimized by setting excitation and emission polarizers crossed at 90°.
FRET was measured using the acceptor normalization method (Clegg,
1992), in which an extracted acceptor spectrum FA(n1,n¢) (excitation at
n¢ = 590 nm for Alexa 594, emission at n1) is normalized to a second
spectrum [F(n1,n")] from the same sample excited at a wavelength
(n" = 655 nm for Alexa 647) at which only the acceptor is excited, with
emission at n2. This gives the acceptor ratio (ratio)A = FA(n1,n¢)/F(n,n").
FRET is considered to be observed if a sample has a (ratio)A value in
excess of that recorded for a control sample having only the acceptor
¯uorophore. Fluorescence intensities were quanti®ed by integrating
emission spectra over the interval from 665 to 700 nm. The extracted
acceptor spectrum was obtained by using regression to ®t the spectrum of
a donor-only control sample to that part of the sample spectrum which has
only donor ¯uorescence (600±620 nm). Subtracting the scaled donor
spectrum from the sample spectrum was used to calculate the extracted
acceptor spectrum.
MALDI-TOF analysis
The band of interest was excised from the gel, and the protein trapped
within the gel was reduced and alkylated and then digested with trypsin
M-LDH as a sarcolemmal KATP channel subunit
(12.5 ng/ml) overnight. The peptides were extracted, ®rst, by the addition
of 25 mM ammonium bicarbonate, followed after a 10 min incubation by
an equal volume of acetonitrile, and then by two extractions with 5%
formic acid and acetonitrile. The pooled extracts were concentrated to
near dryness in vacuo and redissolved in 10% formic acid. Half of the
sample was desalted and concentrated using a micro C18 column (0.2 ml
ZapTip; Millipore) according to the manufacturer's instructions. The
peptides were eluted directly from the tip onto the target in 1.5 ml
a-cyano-4-hydroxycinnamic acid (10 mg/ml) in 75% acetonitrile/25%
formic acid (10%). Spectra were obtained on a Micromass TofSpec 2E
instrument (Micromass, Manchester, UK) equiped with a 337 nm laser
and operated in re¯ectron mode. Three hundred shots were combined and
data were calibrated against a mixture of known peptides. Peptide masses
were searched against the Swiss-Prot/TREMBL databases using the
ExPASy PeptIdent program and against the NCBI database using the MSFit search program (USCF Mass Spectrometry Facility). The searches
were limited to rodent proteins and included an appropriate mass range
limit (Shevchenko et al., 1996).
LDH assay
LDH activity was determined in a reagent solution containg 0.2 M
Tris±HCl (100 ml), 6.6 M NADH (1 ml), 30 mM sodium pyruvate (10 ml);
pH was adjusted to 7.3 at 25°C. LDH activity was measured using a
spectrophotometer (Unicam UV2 Spec) set at wavelength 340 nm on
pellets (20 ml) dissolved in PBS (total volume was 100 ml) and put in
reagent solution (Tris±HCl 1.4 ml, NADH 50 ml, sodium pyruvate 50 ml).
Following 5 min incubation of the reagent solution in the spectrophotometer (to achieve temperature equilibration) pellets were added and
the absorbance at 340 nm was measured every min until steady-state is
reached (steady-state was de®ned as steady reading of absorbance in ®ve
consecutive measurements). The reaction velocity was determined by a
decrease in absorbance at 340 nm, resulting from the oxidation of NADH
indicative of LDH activity.
Patch±clamp electrophysiology
To monitor on-line behaviour of single channel molecules, the gigaohm
seal patch±clamp technique was applied in the inside-out con®guration
(Jovanovic and JovanovicÂ, 2001b). Cells were superfused with (in mM):
KCl 140, MgCl2 1, EGTA 5, HEPES±KOH 5 (pH 7.4). Fire-polished
pipettes, coated with Sylgard (resistance 5±7 MW), were ®lled with (in
mM): KCl 140, CaCl2 1, MgCl2 1, HEPES±KOH 5 (pH 7.3). Recordings
were made at room temperature (22°C), using a patch±clamp ampli®er
(Axopatch-200B). Single-channel activity was monitored on-line and
stored on a PC. Data were reproduced, low-pass ®ltered at 1 KHz (±3 dB)
and sampled at 100 ms rate for further analysis with the pClamp8
software. For whole-cell electrophysiology applied on cardiomyocytes
cells were superfused with Tyrode solution (in mM: NaCl 136.5, KCl 5.4,
CaCl2 1.8, MgCl2 0.53, glucose 5.5, HEPES±NaOH 5.5; pH 7.4). Pipettes
(resistance 3±5 MW), were ®lled with (in mM): KCl 140, MgCl2 1, ATP
3, HEPES±KOH 5 (pH 7.3). The whole-cell K+ current in untransfected or
transfected A549 cells (selection of the transfected cells was based on the
GFP ¯uorescence) was measured using whole-cell perforated patch±
clamp technique with essentially the same pipette solution as above,
except ATP was omitted and amphotericin B (240 mg/ml; Sigma) was
added (see Jovanovic et al., 2001). Also, during ischemia, Tyrode
solution was modi®ed as described below (see section Ischemia). For all
cells monitored, the membrane potential was normally held at ±40 mV
and the currents evoked by a series of 400 ms depolarizing and
hyperpolarizing current steps (±100 to +80 mV in 20 mV steps) recorded
directly to hard disk using an Axopatch-200B ampli®er, Digidata-1321
interface and pClamp8 software (Axon Instruments, Inc., Forster City,
CA). The capacitance compensation was adjusted to null the additional
whole-cell capacitative current. The slow capacitance component
measured by this procedure was used as an approximation of the cell
surface area and allowed normalization of current amplitude (i.e. current
density). Currents were low pass ®ltered at 2 kHz and sampled at 100-ms
intervals. When the effect of LDH substrates on whole-cell K+ current
was assessed either NADH (20 mM), pyruvate (20 mM), NADH plus
pyruvate (20 mM each), NAD (20 mM), lactate (20 mM) or NAD plus
pyruvate (20 mM each) was added into the pipette solution (Crawford
et al., 2002).
Digital epi¯uorescent microscopy
A549 cells (transfected cells were selected based on GFP ¯uorescence)
were superfused with Tyrode solution, and loaded with the esteri®ed form
of the Ca2+-sensitive ¯uorescent probe Fura-2 (Fura-2AM, dissolved in
dimethyl sulfoxide plus pluronic acid; Molecular Probes). Cells were
imaged using a digital epi¯uorescence imaging system coupled to an
inverted microscope (Image Solutions, Standish, UK). A mercury lamp
served as a source of light to excite Fura-2AM at 340 and 380 nm.
Fluorescence emitted at 520 nm was captured, after crossing dichroic
mirrors, by an intensi®ed charge coupled device camera, and digitized
using imaging software. An estimate of the cytosolic Ca2+ concentration,
as a function of Fura-2 ¯uorescence, was calculated according to the
equation: [Ca2+] = (R ± Rmin/Rmax ± R)Kdb, where R is the ¯uorescence
ratio recorded from the cell, Rmin and Rmax the minimal and maximal
¯uorescence ratio, Kd the dissociation constant of the dye (236 nM), and b
the ratio of minimum to maximum ¯uorescence at 380 nm (Ranki et al.,
2001).
Ischemia
Untransfected and transfected A549 cells were exposed to ischemia
combining hypoxia with modi®ed Tyrode solution (ischemic solution).
The ischemic solution (solution otherwise similar to Tyrode solution,
contained slightly increased K+ concentration, 16 mM, pH was reduced to
6.5, and glucose was omitted) was continuously bubbled with 100% argon
and the exchange of O2 between solution in the chamber and air was
prevented by a nitrogen jet. The PO2 under these conditions was
~20 mmHg. The duration of ischemic perfusion was 10 min (Ranki et al.,
2001).
Statistical analysis
Data are presented as mean 6 SEM, with n representing the number of
patched/imaged cells or examined hearts. Mean values between two
groups were compared by the paired or unpaired Student's t-test or rank
tests where appropriate. Mean values between more then two groups were
compared by the one-way or one-way rank ANOVA. All statistical tests
were carried out using using the SigmaStat program (Jandel Scienti®c).
P <0.05 was considered statistically signi®cant.
Acknowledgements
We thank S.Seino (Chiba University) for the Kir6.2 gene, Y.Kurachi
(Osaka University) for the SUR2A gene. We are grateful to C.H.Booting
(University of St Andrews) and D.M.J.Lilley (University of Dundee) for
MALDI-TOF and FRET analysis, respectively. This research was
supported by grants from the Anonymous Trust, BBSRC, British Heart
Foundation, MRC, National Heart Research Fund, TENOVUS and the
Wellcome Trust.
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Received January 30, 2002; revised May 28, 2002;
accepted June 3, 2002
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